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dc.contributor.authorAlagesan, Senthilkumar
dc.description.abstractAllogeneic mesenchymal stem cells (allo-MSC) are a promising “off-the-shelf” therapy and are often cited as failing to elicit anti-donor immune responses. Intra-muscular (IM) administration may be the optimal route for allo-MSC therapy in heart disease and limb ischemia. In the project described in this thesis, anti-donor cellular and humoral immune responses were compared in fully MHC-mismatched mice following single or repeated IM injections of allo-MSC. As positive and negative controls, additional groups of mice received IM injections of allogeneic splenocytes (allo-splenocyte), of syngeneic MSC (syn-MSC) or of vehicle alone. A panel of assay techniques was developed to assess anti-donor immune responses in secondary lymphoid organs including multi-colour flow cytometry panels, dendritic cell (DC)-stimulated mixed lymphocyte reactions (MLRs), cytotoxicity assays and assays of donor-specific IgG antibody. Analyses of anti-donor T cell responses 1 week following the final IM injections demonstrated donor-specific hypo-responsiveness in recipients of repeated (but not single) injections of allo-MSC. In contrast, repeated allo-splenocyte injection was associated with enhanced anti-donor T cell responses. The donor-specific hypo-responsiveness associated with repeated IM allo-MSC was present in both CD4+ and CD8+ T cell compartments. It was not accompanied by expansion of CD4+/FOXP3+ regulatory T cells but was associated with increased production of interferon gamma and interleukin 10 in donor antigen-stimulated MLRs as well as with increased numbers of CD8+/CD11c+ T cells and alterations to the myeloid and natural killer cell/natural kill T cell compartments in the spleen. Analyses of humoral (B cell) responses 1 week following the final injections revealed that recipients of single and repeated IM injections of allo-MSC consistently developed anti-donor IgG antibodies. The donor-specific IgG antibodies of allo-MSC recipients were of similar titres to those observed in recipients of allo-splencoyte injections but had higher ratio of IgG1 to IgG2a isotype. Nonetheless, anti-donor IgG induced by single or repeated IM injection of allo-MSC supported complement-mediated lysis of donor cells, indicating potential to mediate rejection in vivo. In additional experiments involving mice with induced hind limb ischaemia, it was shown that the development anti-donor IgG following IM injection of allo-MSC could be prevented by a short course of the T cell-specific immunosuppressive drug tacrolimus. Taken together, the results from this project provide new insights into the immunogenicity of allo-MSC delivered by the IM route on a single or multiple occasions. Specifically, it was found that IM injection of allo-MSC consistently induced donor-specific T cell and B cell (antibody) immune responses – likely via indirect alloantigen presentation. Importantly, repeated IM administration of allo-MSC resulted in the emergence of donor-specific T cell hypo-responsiveness which we propose may reflect a multi-faceted anti-inflammatory response to MSC-delivered donor antigen that is dependent on an initial “priming” exposure. Also of importance to future clinical application, IM injection of allo-MSC proved to be a potent inducer of IgG1-predominant anti-donor antibody with potential to mediate donor cell lysis. The findings highlight the fact that allo-MSC are, in fact, inherently immunogenic when delivered intramuscularly and that harnessing their tolerogenic potential may, paradoxically, require an initial sensitising phase. These complex interactions between allo-MSC and the recipient immune system should be considered and studied in more detail in human participants in clinical trials involving Allo-MSC administration.en_IE
dc.subjectRegenerative medicineen_IE
dc.subjectStem cellsen_IE
dc.titleImmunogenicity of allogeneic mesenchymal stem cellsen_IE
dc.local.noteThe research work carried out in this thesis focuses on safety (immune response) and associated therapeutic values of a type of stem cells called "Mesenchymal Stem Cells (MSC)" for inflammatory disease. In general, transplantation of any organ or cells from one individual to a genetically unrelated patients (allogeneic transplant) will result in destructive immune response which might even lead to death. However, previous research work shows that MSC don't induce such immune responses because of their inherent biological properties. Nonetheless, no work has been done to exclusively study the immune responses associated with use of allogeneic MSC. The body of research work detailed in this thesis embarked to fully understand the complicated immune response associated with "off-the-shelf" allogeneic MSC therapy. This project has great implications towards clinical development of allogeneic MSC therapy for various diseases such as heart disease and limb ischemia.en_IE

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